Heterotrimeric guanine nucleotide binding proteins (G proteins), composed of Galpha and Gbeta/gamma subunits, transduce signals across cell membranes from receptors to a variety of effectors that include adenylyl cyclases, phosphodiesterases, phospholipases and ion channels. Receptor-stimulation causes Galpha to exchange GTP for GDP and dissociation from Gbeta/gamma. Both subunits interact with effectors until Galpha hydrolyzes GTP to GDP. Hormone-receptor interactions are very specific, but many receptor-G protein interactions are less specific. Reconstitution studies with G proteins, receptors and effectors show that many Galpha subunits can couple to the same receptor and effector. Since multiple G-protein-coupled signaling pathways exist in every eukaryotic cell, there is the potential for crosstalk between signaling pathways. A proposal is that localizing G proteins in specific membrane domains is an important mechanism for signal specificity. This proposal will use mutant Galpha subunits (some already characterized and new one to be made based on crystal structures) to address questions of how Galpha attaches to the membrane, and localizes in specific membrane domains. The interactions of Galpha subunits with the membrane vary among Galpha families. Using in vitro assays and transient transfections, mutations at both the N- and C-termini of Galpha-o, Galpha-s and Galpha-q will be used to identify regions important to membrane binding that are in addition to known roles from lipids and interactions with beta/gamma. With these techniques, amino acids 11-14 of Galpha-o have been found to contribute to membrane binding through an unidentified membrane protein(s). The targeting of Galpha to specific membrane domains is being studied in MDCK cells (polarized epithelia). To follow Galpha in these cells, stable cell lines expressing Galpha-o (not normally expressed) and epitope tagged Galpha subunits are being established and characterized by immunofluorescence and confocal microscopy. Galpha-o localizes to the lateral membrane and overlaps with the endogenous Galpha-i2 and ZO-1 (tight junction (TJ) protein). Mutant Galpha-o subunits and chimeras of Galpha-o and Galpha-s (both apical and basolateral localization) will be used to identify regions important for specific membrane targeting. The pathway(s) of targeting will be established for Galpha-o and endogenous Galpha i2, Galpha-s, and Galpha-q by pulse chase labeling and selective membrane biotinylation. Expression of activated Galpha-o (Q205L) also localizes to the basolateral membrane and causes accelerated formation of TJs using the Ca+2 switch model of TJ biogenesis. A role for Galpha subunits in TJ biogenesis will be further characterized with stable cell lines expressing wildtype and activated Galpha subunits. Accurate signaling is critical for all cells, and signaling pathways are often disrupted after ischemic tissue injury. TJ formation is critical in developing epithelial tissues and during recovery from ischemia (acute tubular necrosis). These studies may give new insights to an understanding of signal specificity in all cells, and may permit the development of strategies to prevent or correct aberrant signaling in diseased or injured tissues.